New Ethylated Derivatives of Sulfur- and Nitrogen-Containing Artifacts from Tenodera sinensis Egg Pod and Their Anti-Renal Fibrosis

Three pairs of enantiomers and one achiral molecule that are new ethylated derivatives of sulfur and nitrogen-containing compounds named mantidisamides E–H (1–4), along with twenty known ones (5–24), were derived from the ethanol extract of Tenodera sinensis Saussure. The structures of these new compounds and their absolute configurations were assigned on the basis of spectroscopic analyses and computational methods. The assessment of activities in NRK-52e cells induced by TGF-β1 demonstrated that the previously undescribed compounds 1 and 2 exhibited a significant capacity to inhibit the expression of proteins (fibronectin, collagen I, and α-SMA). Moreover, the biological activity of these compounds was found to increase with rising concentrations. Notably, compounds 1–4 should be artifacts; however, undescribed compounds 1 and 2, which possessed obvious biological activity, might be attractive for chemists and biologists due to the potential for more detailed exploration of their properties. It is worth mentioning that compounds 1 and 2 remain novel structures even in the absence of the ethoxy group.


Introduction
For centuries, it has been recognized that insects and their products serve as valuable sources of sustenance and medicine, owing to their nutritive properties, diverse chemical compositions, and abundance.Our present research endeavors are focused on investigating the natural substances obtained from Mantidis ootheca, which originated from three species of Mantis: Tenodera sinensis Saussure, Statilia maculate (Thurlberg), and Hierodula patellifera (Serville) [1].The Mantidis ootheca is a very common insect that is mainly distributed in Guangxi, Yunnan, and Liaoning provinces of China, and its potential for therapeutic use in the treatment of kidney diseases has been noted [2,3].In our previous studies, we have reported some interesting small molecules that exhibited COX-2 inhibitory and anti-renal fibrosis activities [4,5].In our quest for a more profound comprehension of the chemical composition and biological activity of Mantidis ootheca, we kept on conducting the study on the specified insect in the title, which resulted in the discovery of three pairs and one previously undescribed compound containing sulfur and nitrogen atoms (Figure 1).Additionally, we have identified twenty previously known compounds (Figure S1).Their structures, including absolute configurations, were determined through a combination of spectroscopic and computational methods.Moreover, in order to assess the biological effects of the undescribed compounds, we investigated their possible anti-fibrotic properties, a pathology characterized by the abnormal accumulation of extracellular matrix (ECM) proteins within the renal system. 1 2 of 14 combination of spectroscopic and computational methods.Moreover, in order to assess the biological effects of the undescribed compounds, we investigated their possible antifibrotic properties, a pathology characterized by the abnormal accumulation of extracellular matrix (ECM) proteins within the renal system.
Compound 3 ((±) mantidisamide G), a colorless gum, has the molecular formula of C13H16N2O3S by analysis of its negative HRESIMS at m/z 279.0808 [M-H] − (calcd for C13H15N2O3S, 279.0809), with 7 degrees of unsaturation.The 13 C NMR combined with the  1).The 13 C NMR and DEPT spectra of 2 (Table 1) indicate 14 carbon signals, comprising two methyls, three methylenes, three methines (one nitrogen or oxygen-bearing and two olefinic), and six nonprotonated carbons (two carbonyls and four olefinic).A detailed comparison of 1D NMR data between compounds 1 and 2 indicated that both of them have the same basic structural fragment of benzothiazide.The  2) show 13 carbon signals attributed to 2 methyls, 2 methylenes, 4 methines, and 5 nonprotonated carbons (containing one carbonyl).A detailed analysis of the NMR data suggested that the structure of 3 is similar to that of polyrhadopamine D [6], except for an additional ethoxy group at C-7 in 3, evidenced by the 1 H-1 H COSY correlation of H 2 -12 (δ H 3.44)/H 3 -13 (δ H 1.20) (Figure 2), and the HMBC correlation of H 2 -12/C-7 (δ C 81.5) (Figure 2).Thus, the planar structure of 3 was identified.Two enantiomers were gained from 3 by means of chiral HPLC purification, and their absolute configurations were assigned as 7S for (−)-3 and 7R for (+)-3 by comparing the calculated ECD spectra with the experimental ones (Figure 3).2) reveals that they have similar structures differing in that the ethoxy group in 4 is located at C-6, which was demonstrated by the HMBC correlation of H-2 (δ H 6.67)/C-6 (δ C 157.2) and H 2 -12 (δ H 4.11)/C-6 (Figure 2).Therefore, the planar structure of 4 was assigned and named mantidisamide H.
Of note, compounds 1-4 all contain an ethoxyl group in the structures.Normally, this change could be considered to be artificial in nature during extraction or isolation procedures.In this study, we proposed that 1-4 are also artifacts during the extraction process since EtOH was used for extraction.In spite of this, we still changed the alterative solvents, including acetone and MeOH, to extract from raw materials under mild conditions.Then, we made an effort to examine whether 1-4 are present or not by HPLC-MS experiments.As expected, 1-4 were not detectable in the above extracts (Figures S44-S47), suggesting that they should be artificial products.Despite this, it will have no negative influence to add the structural or biological diversity of such types of structures where the biological activity has been confirmed by the following experiments in this study (see below).More significantly, it has been determined that the structures of compounds 1 and 2 are novel even without the ethoxy group.
In order to understand the sources of these molecules, the biosynthetic pathways of compounds 1-7 have been discussed (Scheme 1).The proposed biosynthetic pathway of the mantidisamides commenced with tyrosine, which was converted to DOPA, followed by norepinephrine (NE) [26], 1,4-benzothiazine (BT) or benzothiazine carboxylic acid (BTCA) [27], and N-(3,4-dihydroxyphenethyl) acetamide (NDHPA) through several enzymatic transformations.The unstable intermediates, BT and BTCA, may possibly lead to the formation of 3-oxo-3,4-dihydro-1,4-benzothiazine (ODHBT), and the benzothiazole (BZ).Furthermore, ODHBT decarboxylated followed by acetylation with acetyl-CoA led to the formation of ODHBT-CoA.Finally, ODHBT-CoA was then converted to compound 2 through oxidation by a hydroxylase and subsequently ethylated.BZ decarboxylated and acylated to afford 5. Compound 5 transformed into a non-isolated precursor A2 through oxidation, prone to dehydration under acidic conditions or when extracted as mixtures with acidic co-metabolites to produce an aziridine intermediate C, which was a nucleophilic addition with EtOH at C-7 and C-6 positions to generate the compounds 3 and 4, respectively.In addition, racemic artifacts formed due to nucleophile (EtOH) attacks from the top side and bottom side at the planner carbocation position; in particular, EtOH attacked from both sides at C-7 positions of aziridine intermediate C to generate the racemic compound 3. NE was acylated with acetyl-CoA and followed by ethylation to obtain compound 6.The hydroxyl group in NDHPA was substituted with a chlorine atom to form 7. The biosynthesis of compound 1 began with quinone, which was converted to 5-S-cysteinyl quinone (CQ) via a rapid addition reaction.CQ underwent intramolecular cyclization by condensation of the pendant amine, affording 2H-1,4-benzothiazine (DHCQ) [28].Furthermore, decarboxylation and oxidation introduce a hydroxy group to the obtained A1.A1 is accelerated to dehydration to produce the achiral thionium intermediate B, which can be quenched by EtOH to deliver racemic compounds 1 and 2. Finally, the proposed biosynthesis of compounds 3 and 4 formed compound 5, suggesting that the mild conditions used during the extraction process were favorable for selective mono ethylation.Hence, we conclude that these isolates could be artifacts, and these artifacts obtained under acidic co-metabolites act as a catalyst during the extraction/isolation of the insect T. sinensis.

Biological Activity
Chronic kidney disease (CKD) is commonly associated with renal fibrosis and is regarded as its histological endpoint.Similar to fibrosis in other organs, renal fibrosis is characterized by the pathological deposition of the extracellular matrix (ECM) [29].Multiple factors contribute to the development of fibrosis, and, among them, transforming growth factor-β1 (TGF-β1) plays a crucial role in driving this pathological process [30].Therefore, the anti-renal fibrosis activities of the newfound compounds in NRK-52E cells induced by TGF-β1 were evaluated by measuring the levels of ECM proteins such as fibronectin, collagen I, and α-SMA.Initially, the cells' potential cytotoxicity was successfully eliminated through testing cell viability by a CCK-8 assay; the compounds were nontoxic at 20 µM (Figure 4).Subsequently, we proceeded to evaluate the expression of ECM-related proteins, and found that the expression of α-SMA, collagen I, and fibronectin exhibited a notable reduction in response to compounds (±)-1 and (±)-2 (Figure 5).Furthermore, doseresponse studies revealed that the anti-fibrotic activities of compounds (±)-1 (Figure 6A-D) and (±)-2 (Figure 6E-H) appeared to be more prominent with increasing concentrations; even the inhibition of ECM-associated proteins was discernible at 5 µM.

Biological Activity
Chronic kidney disease (CKD) is commonly associated with renal fibrosis and is regarded as its histological endpoint.Similar to fibrosis in other organs, renal fibrosis is characterized by the pathological deposition of the extracellular matrix (ECM) [29].Multiple factors contribute to the development of fibrosis, and, among them, transforming growth factor-β1 (TGF-β1) plays a crucial role in driving this pathological process [30].Therefore, the anti-renal fibrosis activities of the newfound compounds in NRK-52E cells induced by TGF-β1 were evaluated by measuring the levels of ECM proteins such as ficollagen I, and α-SMA.Initially, the cellsʹ potential cytotoxicity was successfully eliminated through testing cell viability by a CCK-8 assay; the compounds were nontoxic at 20 µM (Figure 4).Subsequently, we proceeded to evaluate the expression of ECMrelated proteins, and found that the expression of α-SMA, collagen I, and fibronectin exhibited a notable reduction in response to compounds (±)-1 and (±)-2 (Figure 5).Furthermore, dose-response studies revealed that the anti-fibrotic activities of compounds (±)-1 (Figure 6A-D) and (±)-2 (Figure 6E-H) appeared to be more prominent with increasing concentrations; even the inhibition of ECM-associated proteins was discernible at 5 µM.

Insect Material
The insects were identified as the egg cases of T. sinensis by Prof. Da-Rong Yang at the Kunming Institute of Zoology, Chinese Academy of Sciences, which were purchased from Anshan, Liaoning, China, in March 2021.The voucher specimen (CHYX0647) has been placed in the School of Pharmacy, Shenzhen University Medical School, Shenzhen University, China.

Extraction and Isolation
The insect materials (30.0 kg) were pulverized and then extracted three times with 50% ethanol (200 L × 48 h × 1, 150 L × 48 h × 2) at room temperature.This process resulted in the production of the crude extract, weighing 2.1 kg.Afterwards, the extract was fractionated using a macroporous resin column, and carefully eluted with EtOH/H 2 O (0-100%) to produce six fractions (Fr.A-Fr.F).The comprehensive isolated procedures are included in the Supporting Information.Racemic mixtures of compounds 1-3 were acquired and subsequently purified by chiral HPLC on a Daicel Chiralpak column (IC, 250 mm × 4.6 mm, i.d., 5 µm).  1C NMR data (see Table 2).

ECD Calculations
The ECD calculations were conducted using a similar approach as described in the literature [31].The lowest energy of all the conformers of each compound was given by CONFLEX 7 software, and then the optimized and ECD calculations were carried out based on the mPW1PW91/6-311 + G(d,p) level with the PCM model in methanol for (8R)-1 and the B3LYP/6-31G (d, p) level with PCM in methanol for (8R)-2 and (7R)-3.SpecDis 1.62 and Origin 2019 programs were utilized to analyze the ultimate data.Normal rat kidney proximal tubular epithelial cells (NRK-52e) (Cell Bank of China Science Academy, Shanghai, China) were cultured in 10 cm dishes and maintained in high glucose DMEM (C11995500BT, Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (2094468CP, Gibco, Waltham, MA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin.The cells were incubated at 37 • C in a humidified atmosphere with 5% CO 2 .

Figure 4 .
Figure 4. Assessment of the toxicity of compounds 1-4 in NRK-52e cells.NRK-52e cells were exposed to compounds at 20 µM for 48 h, and the cell viability was assessed by using the CCK-8 assay.n = 6 biologically independent cells.

Figure 4 .
Figure 4. Assessment of the toxicity of compounds 1-4 in NRK-52e cells.NRK-52e cells were exposed to compounds at 20 µM for 48 h, and the cell viability was assessed by using the CCK-8 assay.n = 6 biologically independent cells.

Figure 4 .
Figure 4. Assessment of the toxicity of compounds 1-4 in NRK-52e cells.NRK-52e cells were exposed to compounds at 20 µM for 48 h, and the cell viability was assessed by using the CCK-8 assay.n = 6 biologically independent cells.